The isolation of a specific subset of cells from a heterogeneous population of cells is necessary for a range of research and diagnostic tools. For example, isolation of circulating tumor cells (CTCs) from a buffy coat formed from a patient blood draw has shown clinical relevance. As is known, however, CTCs within the circulation of patients with metastatic cancer are very rare. More specifically, there is approximately one CTC per billion background cells. Further, the prognostically relevant bar for determining overall survival and disease-free progression of a patient is 5 CTCs per 7.5 milliliters (mLs) of whole blood. As such, CTC capture is an isolation method requiring both high sensitivity (5 cells) and high specificity (7.5 billion background cells). In addition, once captured, clinically relevant cellular analyses must be performed on the CTCs downstream of isolation.
While offering the flexibility to perform a wide range of downstream assays, macroscale methods to perform CTC isolation have been found to be unsatisfactory. More specifically, macroscale methods to isolate these types of cells often require long, expensive and laborious procedures that may result in significant sample loss due to wasteful transfer steps or centrifugation and resuspension steps. Capturing rare cells such as CTCs, which occur at frequencies on the order of 5-50 cells per 7.5 mL blood sample, is not feasible using traditional macroscale methods, as the loss of a single cell can represent up to a 20% loss of sample.
In order to overcome cell loss associated with the macroscale methods, heretofore described, microfluidic methods have arisen. Microfluidics offers novel solutions to the challenges of macroscale methods by providing a highly controlled, low-volume platform that can quickly and efficiently isolate cells. Further, microfluidic platforms offer sensitivity and specificity that is unattainable using current macroscale systems. Established microfluidic methods include functionalized micropost arrays, patterned surfaces and microfluidic systems that leverage density or other physical characteristics to isolate cells of interest from non-target cells. By way of example, Beebe et al., United States Patent Application No. 20110213133 discloses a device and a method for facilitating extraction of a fraction from a biological sample. The biological sample includes non-desired material and a fraction-bound solid phase substrate. The device includes an input zone for receiving the biological sample therein and a second zone for receiving an isolation buffer therein. An output zone receives a reagent therein. A force is movable between a first position adjacent the input zone and a second position adjacent the output zone. The force urges the fraction-bound solid phase substrate from the input zone, through the second zone and into the output zone.
While functional for its intended purpose, the device and method disclosed in the Beebe et al., '133 publication has certain limitations. For example, when the biological sample contains large particulates, debris, precipitates, or other cells that settle out of solution, the efficiency of the recovery and the overall purity of the fraction-bound solid phase substrate decreases as a result of non-desired material impeding the operational path of the fraction-bound solid phase substrate.
Therefore, it is a primary object and feature of the present invention to provide a device and a method isolating a fraction from a biological sample.
It is a further object and feature of the present invention to provide a device and a method for isolating a fraction from a biological sample that is simpler and more efficient than prior devices and methods.
It is a still further object and feature of the present invention to provide a device and a method for isolating a fraction from a biological sample without the significant sample loss associated with prior methods.
In accordance with the present invention, a device is provided for isolating a fraction in a biological sample. The fraction is bound to solid phase substrate to define a fraction-bound solid phase substrate. The device includes an input zone for receiving the biological sample therein and an isolation zone for receiving an isolation fluid therein. A force, generally perpendicular to gravity, is movable between a first position adjacent the input zone and a second position adjacent the isolation zone. The force captures the fraction-bound solid phase substrate such that the fraction-bound solid phase substrate moves from the input zone to the isolation zone in response to the force moving from the first position to the second position.
The input zone is partially defined by a lower surface lying in a first plane and wherein the device further comprising a passage having a input communicating with the input zone and an output communicating with the isolation zone. The passage is partially defined by first and second walls. The first and second side walls of the passage converge from the input to the output thereof. The passage extends along an axis. The axis is vertically spaced from the first plane. The isolation zone is partially defined by a lower surface lying in a second plane, the second plane being between the first plane and the axis. It is contemplated for the force to be a magnetic field. Further, it is contemplated for the force to move from the first position to the second position along a path transverse to gravity.
In accordance with a further aspect of the present invention, a device is provided for isolating a fraction in a biological sample. The fraction is bound to a solid phase substrate to define a fraction-bound solid phase substrate. The device includes an input zone for receiving the biological sample therein. The input zone is partially defined by a lower surface lying in a first plane. An isolation zone receives an isolation fluid therein. The isolation zone is partially defined by a lower surface lying in a second plane. A passage extends along an axis and has an input communicating with the input zone and an output communicating with the isolation zone. A force captures the fraction-bound solid phase substrate. The force is generally normal to gravity and is movable between a first position adjacent the input zone and a second position adjacent the isolation zone. The captured fraction-bound solid phase substrate moves from the input zone to the isolation zone in response to the force moving from the first position to the second position.
The passage is partially defined by first and second walls. The first and second side walls converge from the input to the output thereof. The axis of the passage is vertically spaced from the first plane and the second plane is between the first plane and the axis. It is contemplated for the force to be a magnetic field. Further, it is contemplated for the force to move from the first position to the second position along a path transverse to gravity and to the force.
In accordance with a still further aspect of the present invention, a method is provided of isolating a fraction in a biological sample. The method includes the step of providing a biological sample including a fraction-bound solid phase substrate and biological material in an input well. The input well is partially defined by a lower surface lying in a first plane. The fraction-bound solid phase substrate is captured with a force so as to maintain the fraction-bound solid phase substrate at a location above the lower surface of the input well. The biological material is allowed to settle towards the lower surface of the input well and the fraction-bound solid phase substrate is drawn into an isolation well through a passage with the force. The passage extends along an axis vertically spaced above the first plane.
It is contemplated for the force to be generally normal to gravity and to be a magnetic field. The force travels along a path to draw the fraction-bound solid phase substrate from the input well into the isolation well. The path is transverse to gravity. The passage has an input communicating with the input zone and an output communicating with the isolation zone. The passage is partially defined by first and second walls. The first and second side walls converge from the input to the output thereof. The isolation zone is partially defined by a lower surface lying in a second plane. The second plane is between the first plane and the axis.